Immunoassays for specific antigens are powerful tools for clinical diagnostics and for various molecular and cellular analyses. The instant invention is directed at enhancing the sensitivity of immunoassays by employing variant DNA as an amplifiable reporter molecule.
The sensitivity of conventional immunoassays is in practice governed for a particular antibody-analyte interaction by the value of the equilibrium constant, which is normally referred to as the dissociation constant. The equilibrium or dissociation constant reflects the amount of antibody-analyte complex formed at given concentrations of reactants. Typical values for the dissociation constant are found in the range of 10.sup.-7 M to 10.sup.-12 M. For example, at an antibody concentration equal to the dissociation constant, approximately 50% of a trace analyte will be complexed with the antibody at equilibrium.
Efforts to maximize sensitivity for immunoassays have included attaching labels, such as, enzymes, to antibodies such that the number of antibody-analyte complexes which can be detected is maximized. The ultimate goal in such an approach is to be able to detect a single analyte-antibody complex.
An article by Chu et al., [Nucleic Acids Research, 14: 5591 (1986)], concerns the use of midivariant RNA as an amplifiable reporter to enhance the sensitivity of bioassays. Midivariant RNA serves as a template for its own replication which is catalyzed by QB replicase. [Miele et al., J. Mol. Biol., 171: 281 (1983).] A single molecule of midivariant RNA theoretically could be amplified by QB replicase to give sufficient copies to enable detection by conventional radioactive and non-radioactive techniques. Chu et al., supra state at page 5602 that "the theoretical sensitivity of an assay system that uses . . . [midivariant RNA as a] replicable reporter should be close to one molecule of target." However Chu et al. go on to state that they "anticipate that the detection limits that can be reached in practice will be determined by irreducible, nonspecific adsorption of the receptor to the sample."
That problem of nonspecific adsorption of a receptor carrying such a midivariant RNA label could be a serious drawback because of the efficiency with which the midivariant RNA template may be replicated. Midivariant RNA templates from a relatively few nonspecifically adsorbed receptors could result in an extremely high background signal which could obscure the signal from specifically bound receptors. The sensitivity of such an assay using midivariant RNA as an amplifiable reporter would be therefor limited.
This invention solves that problem by using variant DNAs, rather than midivariant RNA, as templates for RNA synthesis with appropriate RNA-dependent RNA polymerases, such as, QB replicase. As variant DNA templates, such as, nanovariant and midivariant DNA templates, do not replicate as efficiently as their RNA counterparts, such DNA templates overcome the potential problem of extremely high background signal that could occur if variant RNA templates were used.
The immunoassay methodology of this invention employs variant DNA as an amplifiable label conjugated to an antibody or biologically active antibody fragment. The advantages for the use of the amplifiable DNA templates of this invention over their RNA counterparts as labels for antibodies are several. First, current methods of synthesizing DNA oligonucleotides are further advanced than for their RNA counterparts. Second, DNA is inherently more stable than RNA. Also, as indicated above, the relative inefficiency of the replication of the DNA templates can be used effectively to mask the potential signal generated from non-specific binding of the antibody conjugates, and thus, the assays of this invention should be more sensitive than assays employing corresponding RNA template conjugates.